Paper - Primary neuromeres and head segmentation (1922)
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Primary Neuromeres and Head Segmentation
Horace W. Stunkard
New York University
The problem of the segmentation of the vertebrate head, old as Oken and Goethe, has possibly attracted as much interest and incited as much investigation as any other one question in vertebrate morphology. The first investigators advanced a theory based upon superficial external features the sutures of the skull. Subsequent workers have investigated every structure enclosed within the skull, with the hope that light may be thrown upon the obscurity and uncertainty enveloping the evolution of the head. This complex, intricate structure manifests evidences of past ages; remnants and vestiges of the long period of developmental history still persist, but the character of the evidence, the complications, omissions, and reversals have baffled all attempts at solution.
Huxley ('58) overthrew the vertebral theory of the skull, Balfour (78) introduced mesodermal head cavities as criteria of segmentation and clues to the number and relationship of the cephalic somites, and Gegenbaur ('87) added cranial nerves and visceral arches as segmental criteria. Van Wijhe ('86), ('89) considered the dorsal ganglia of importance and formulated criteria to determine the true segmental nerves. He regarded the olfactory and optic nerves as parts of the brain and not of seg^ mental value.
Von Baer ('28) noticed symmetrical folds in the hindbrain of the chick; Dohrn ('75) related them to the mesodermal somites, and Beraneck ('84) to the cranial nerves. Balfour reported that the first gives rise to the cerebellum, and considered it doubtful whether the other constrictions have any morphological importance. Von Kupffer ('85) observed a 'primary metamerism' in the neural tube of Salamandra atra embryos which appeared before the segmentation of the mesoderm, and Orr ('87), studying the embryology of the lizard Anolis, noticed a number of symmetrical constrictions in the lateral walls of the hindbrain, giving the walls in horizontal section an undulated appearance." Kupffer called these 'medularfalten' and Orr adopted for them the name neuromeres. This author formulated the first criteria for determining the identity of the neuromeres. He described two in the primitive forebrain, one in the midbrain, and six in the hindbrain. McClure ('90), working on embryos of Amblystoma punctatum, Anolis sagroei, and the chick, found "a continuous and symmetrical series of neuromeres increasing in size anteriorly, which extend from the lateral walls of the embryonic brain, throughout the entire length of the neuron." He believed that the primary forebrain contained two neuromeres, that the midbrain consisted of two neuromeres, and that the third and fourth nerves were the nerves of these somites. Froriep ('91) found neuromeres prior to the segmentation of the mesoderm, but did not attach any segmental importance to them, and later ('92) decided they were the results of underlying mesoblastic somites. He found the constrictions in the median part of the cephalic plate, while the neural tube is still open, four in Salamandra maculosa and five in Triton cristatus. Waters ('92) confirmed the observations of McClure, and found three segments in the forebrain. Eycleshymer ('95) observed certain markings in the neural folds which might be interpreted as neuromeres, yet he noted that their arrangement was decidedly irregular and the structures were probably due to the action of killing reagents. The transverse markings in the neural plate he regarded as due to the formation of the myomeres.
The tendency to regard the neuromeres as segmental structures reached a definitive stage with the work of Locy ('95). This author reviewed the w^ork on neuromeres exhaustively. He made observations on Squalus acanthias, Amblystoma, Diemyctylus, Rana palustris, Torpedo ocellata, and the chick. In all these forms he described neuromeres in very early stages, as soon as the neural folds are established, and before there is any division of the mesoderm into protovertebrae. In the open neural groove, the neuromeres of the hindbrain are, he stated, merely the more apparent constrictions of a neuromerism that involves the entire neural plate. He traced the neuromeres to the anterior end of the medullar}' groove and those earliest formed without a break into the later stages, identifying them with the neuromeres of the closed neural tube. In the chick he described neuromeres visible in the blastoderm of the twelfth hour of incubation, and stated that this segmentation extends into the primitive streak. In Aiiiblystoma, at the stage with a broadly expanded neural plate and widely open neural groove, he found "the neural folds divided throughout their length into a series of segments with no especial distinguishing features between those of the head and those of the body region. The median plate included between the neural ridges is smooth at this stage : at a shghtly later period, however, while the groove is still v.'idely open, the median plate exhibits very faint transverse markings." He pointed out that these median di\dsions do not correspond with those in the neural ridges, and he attached no morphological significance to them. He claimed that in all the forms studied "The cells in the neural segments are characteristically arranged, even in the earliest stages, and their arrangement and structure would indicate that they are definite differentiations of cell areas, not merely mechanical undulations." Locy summarized his work on neuromeres by stating that they cannot be artifacts, that they arise before there is any segmental division of the mesoderm, and so cannot be dependent upon the latter. He concluded that neuromeric segmentation is more primitive than mesodermic segmentation, and for this reason msiy well serve as a basis for the study of the segmentation of the head.
Neal ('98) was unable to verify Locy's statements in Squalus. He found the edges of the plate slightly and irregularly lobed, but the lobes on the opposite margins of the plate did not correspond either in number or position, nor did they show any definite relation to the mesodermal somites. Regarding these 'segments' as the results of unequal growth along the margin of the neural plate, he contended that it is obviously not necessary to regard such irregularities of the edge of a rapidly expanding plate of tissue as of morphological importance. A disassociation of cells or rapid proliferation of cells, which certainl}^ does occur in this region, would lead to such phenomena." Neal found it impossible to trace definite segments into the later stages, for in these stages, before the closure of the neural tube, in the majority of specimens little or no evidence of segmentation along the cephalic plate could be seen. In Squalus acanthias he found the posterior boundary of the cephalic plate coincides with the posterior boundary of encephalomere VI, opposite which the auditory invagination takes place. Showing discrepancies in Locy's statements regarding the position of the auditory vesicle and the posterior limit of the cephalic plate, Neal says, ^'I can see no escape from the conclusion that he (Locy) has not traced neural segments accurately up to the time they form neuromeres." Furthermore, Neal warned against formulating conclusions from observation of a single organ system and applying them to the phylogenesis of the vertebrate head. He contended that primitively there existed a correspondence between neuromerism, mesomerism and branchiomerism, and the problem of phyletic cephalogenesis is to explain the present lack of correspondence. In Squalus he found five mesomeres alternating with six neuromeres in the otic and preotic region.
Hill (1900), working on Salmo and chick embryos, confirmed the statements of Locy. He reported complete agreement as regards the number and position of the neural segments in the trout and chick embryos. The forebrain has three and the midbrain two segments which, in the earhest stages, do not differ in any essential features from those of the medulla. They antedate the historic divisions, forebrain and midbrain, and precede the optic evaginations. The primary neuromeres were constantly and normally present in the early stages of all the embryos examined by him. Speaking of the external and corresponding internal constrictions which separate the segments, he says that in the early stages these grooves encircle the encephalon, but in later stages the primary segmentation is confined to its base and lateral walls, owing to the neural expansion and the appearance in the dorsal region of a thin roof. He pointed out that in the position occupied by the third, fifth, and sixth segmental grooves, deep internal constrictions appear that form the posterior limits respectively of the forebrain, midbrain, and cerebellum; that all the primitive grooves disappear during embryonic growth, those of the forebrain first, those of the midbrain second, and lastly those of the hindbrain. In the chick, when the neural folds close to form the neural tube, the walls of the latter expand, not uniformly, but intrasegmentally, and the position of the internal grooves is thus passively elevated upon crests. Contrary to the statement of Locy, he found that in younger embryonic stages of the chick and also of the trout, the histology is very simple, the radial arrangement of cells is absent, the nuclei do not recede intrasegmentally from the inner surface of the brain but are uniformly distributed. In these stages he reports that the only criteria by which he has counted neural segments were external and corresponding internal grooves. Concerning the value of various segmental criteria. Hill stated that mesomeres are found only in elasmobranchs, amphibians, and reptiles, and added that in elasmobranchs, the only group in which their development has been traced, their study has led to a greater divergence of opinion and more conflicting views than is generally supposed. He dismissed branchiomeres with a quotation from IMinot that the gill clefts are not segmental and concluded therefore that the branchial nerves are not in segmental order. He argued that Neal's ('98) conclusions were based on negative evidence and that he had observed the segments where Neal failed to find them.
Johnston ('05) accepted the number of neuromeres described by Locy as necessary to account for all the nerves and sense organs connected with the brain, and stated that observations, then incomplete, on embryos of Amblystoma punctatum seemed to confirm Locy's work. He contended that the nervous system, acting in the role of a connecting and coordinating system, might well act as a key for the interpretation of the facts secured by a study of the other structures.
Von Kupffer ('06) reviewed the work of Locy and Hill and maintained that the question is still unanswered. Concerning Hill's work on the neuromeres of the chick, he says, Mit einiger Ueberraschung werden wohl allgemein die Abbildungen aufgenommen worden sein, mit denen Hill seine Beobachtungen iiber die Primaren Neuromeren bein Hiihnchen belegt. Es macht den Eindruck, als wenn das subjective Moment die Fiihrung des Zeichenstiftes doch wohl etwas zu stark beeinflusst hatte"; and (p. 248) ^'Ich kann diese Angaben. mangels gleich ausdehnter Beobachtungen, zwar nicht bestatigen aber ich will sie nicht beanstanden." Neal ('14) translated 'mangels' in this last sentence to mean 4n spite of,' which somewhat alters the original meaning.
Filatoff ('07) argued that neuromeres are mechanical results, due to growth in a restricted space. He rejected Hill's contention that neuromeres are the chief and only certain criteria upon which to build a judgment concerning the primitive metamerism of the head, and agreed with Neal ('98) and Koltzoff ('01) that the proper method by which to attack the problem is to establish an agreement between the neural segments and the somites, nerves, and gill clefts.
Wilson and Hill ('07) could not accept the conclusions of Locy and Hill, and maintained that Hill had not adequately met the contention of Neal ('98).
Belogolowy ('10) maintained that the neuromeres are only form changes of uncertain nature and irregular appearance, possibly the results of mechanical factors, and that they are of most uncertain value as criteria of the segmentation of the head.
Griggs ('10) sought again to establish neuromerism as a basis for determining the segmentation of the head. He described four neuromeres in the procephalic part of the open neural plate of Amblystoma embryos, and in a few specimens of later stages noted neuromeres which appear posterior to the four procephalic lobes, but the history of these posterior neuromeres could not be traced nor their number or arrangement determined. He agreed with Locy that in the early stages the plate and neural crests are net segmented in the same way; he found occasional slight headings or lobes on the neural crests, but did not regard them as of morphological importance. These lateral lobulations he found vary both in number and arrangement and as the neural crests close over the plate all signs of segmentation behind the procephalic lobes disappear. He described three distinct grooves, the anterior and posterior germinal depressions and the 'blastogroove' which appear in the location later occupied by the neural groove. Griggs stated that the primary neuromeres described by Loc}^ were not apparent in any of the embryos which he examined. He concluded that the median transverse grooves separate the true neuromeres, that the first contributes to the formation of the forebrain, the second and third to the formation of the midbrain, and the fourth to the formation of the anterior part of the cerebellum.
Smith ('12) described grooves which appeared very early in the neural place of Cryptobranchus embryos, one regularly antedated the others, and this I believe corresponds to the transverse cephalic groove of Griggs. Anterior to this groove he noted six transitory furrows and posterior to it an undetermined number, but expressed the suspicion that these grooves are connected with the formation of the mesodermal somites. In early stages of the formation of the neural folds he observed occasional transverse grooves, but stated that they are often irregular and bear no definite relation to the segments of the neural plate. He argued that the true segments are to be found between the transverse grooves of the neural plate, and pointed out that in the region of the mesodermal somites the transverse grooves of the plate are in line with the intersomitic grooves and the neuromeres are in line with the somites. He was unable to follow the various structures of the neural plate into the definitive divisions of the einbryonic and adult brain.
Graper ('13) reviewed the literature on neuromeres extensively; he criticised Hill's iiberraschenden und von niemand bestatigten Zeichnungen" and was unable to confirm his observations in the chick.
Neal ('14) argued that the hindbrain neuromeres manifest a segmentation that cannot be explained upon purely mechanical grounds, but contended that the differences in observation and the divergent conclusions of investigators who have examined neuromeres mihtate against the confidence of Locy, Hill, Johnston, Griggs, and others who hold that through the study of neuromerism the primitive segmentation of the head will be ascertained. Quoting Dr. Bashford Dean, he stated that in the forebrain of Bdellostoma there appear two, three, or four neuromeres on one side or the other, never paired; in the midbrain there is any number from one to eight ; while in the hindbrain the number varies from three to twenty-four, differing in number on different sides, a difference of ten having been noted in the right and left sides of the same individual. He added that he had examined hundreds of Squalus embryos in an attempt to confirm Locy's results, and only two or three showed symmetry or regularity in the segmentation of the edges of the neural plate, while the beaded thickenings were not only asymmetrical, but quite variable in different specimens. He maintained that the primary brain vesicles and not their secondary subdivisions are homologous with the hindbrain neuromeres and that the correspondence in number of primar}^ brain vesicles, myotomes, and crania] nerves argues strongly for the metameric value of these structures.
Smith ('14) observed transverse markings in the procephalic plate of Desmognathus fusca embryos which were very transient, varied in position in different individuals, and w^hich he was unable to trace through from one stage to another in living specimens. In the posterior part of the plate the markings were more uniform, persisted longer, and were subject to but slight variation. In some specimens they corresponded closely to the outpocketings in the medullary folds, but not in other individuals. He also described plications in the medullary folds which appeared early and persisted until fused and absorbed in the expanding prosencephalon. These lateral irregularities did not correspond to the median grooves and he ascribed their formation to mechanical factors. He pointed out that it would be easily possible to select from the material a series which would show a uniform development and fate of these foldings, but after the examination of a large number of specimens decided that they had no definite significance or fate. He regarded the folds in the medullary plate as normal, but not constant, and no evidence was found in the cephalic portion of the plate of divisions to which a segmental value should be assigned.
Neal ('18) has admirably summarized the evidence for and against the metameric importance of neuromeres. Adducing evidence from a thorough study of the problem of head development, he contends that the neuromeres of the spinal cord are passive results of the mechanical pressure of the adjacent mesodermic somites ; that the rhombomeres have arisen in correlation with the visceral arches with which they are functionally connected; and that the only structures anterior to the medulla which may h& considered as segmental are the primary forebrain and midbrain segments.
A number of other investigators have worked on different phases of the head-segmentation problem and a more extended review of the literature may be found in the bibliographies of the papers cited here.
It is apparent from the disagreement in the results of former investigators that the nature, number, and significance of the neuromeres are far from determined. While neuromeres have been frequently observed in many animals, and widely discussed, the conception of their value as segmental criteria has been largely developed by Locy, Hill, Johnston, and Griggs. It is extremely difficulb, if not impossible, to correlate the observations and interpretations of the various authors, but the repeated observation as to some kind of division in the neural crests and open neural plate is sufS.cient to warrant further investigation. At the suggestion of Prof. J. S. Kingsley, the writer has studied the early stages in Amblystoma and the chick. The work was begun in 1914 and carried on for two years in the zoological laboratory of the University of Illinois. It was interrupted for two years because of military service, but was continued and completed in the biological laboratory of New York University. The writer wishes here to express to Professor Kingsley his appreciation for the many helpful suggestions received in the course of the stud}'. An attempt was made to determine whether in Amblystoma a segmentation of the neural crests is regularly and uniforml}^ present, and to compare this division with that of the neural plate. Further, to determine, if possible, which, if either, is of metameric significance. The persistent doubt regarding the accuracy of the observations of Locy and Hill on chick embryos makes a reinvestigation and confirmation of their work very desirable.
The study of Amblystoma was made upon several hundred embryos, collected near Champaign-Urbana, Illinois. The entire series of changes involved in the formation and closure of the neural tube was repeatedly observed under the binocular. To make more careful observation, parts of the neural crests and medullary plate were dissected and observed from all angles and with various means of illumination. For material to supplement the study of living specimens, embryos at all stages of development from the wide-open to closed neural tube were killed in various fluids and sections were cut in transverse, frontal, and sagittal planes.
In Amblystoma, as the blastopore narrows to a small oval structure, a distinct longitudinal groove forms anterior to it. In a few specimens the groove appears to extend to the lip of the blastopore, but in the large majority of embryos when the groove is first formed a short distance separates it from the blastopore. In some embryos the groove extends anteriorlj^ and posteriorly in a continuous manner, so that with the closing of the blastopore, it forms the definitive neural groove. In other specimens, however, another faint groove, usually shorter than the first, may appear anterior to it. This observation agrees with that of Griggs ('10), although the appearance of the grooves does not show the regularity or constancy reported by him. The first of these grooves he termed the posterior germinal depression and that anterior to it the anterior germinal depression. The groove formed by the concresence of the lateral lips of the blastopore he called the blastogroove. There is considerable variation in the uniformity and regularity with which these grooves appear, often separate germinal grooves are entirely absent and the neural groove forms without the previous appearance of separate depressions. Griggs stated that it is sometimes impossible to distinguish between the anterior depression, posterior depression, and neural groove. The depressions described by Griggs appear in the same position as the neural groove, and I see no good reason for considering them as anything other than stages in the formation of the neural groove itself. The anterior end of the neural groove is marked by a depression, the anterior pit of Griggs, which becomes deeper, extends anteriorly, and, with only slight variations in the process of development, becomes the infundibulum. Tn these early stages it is sometimes possible to distinguish in the neural groove faint alternating lighter and darker areas, which in a few specimens suggest a segmental condition, but observation of a large number of embryos shows such variation and irregularity as to preclude such an interpretation.
Lateral longitudinal depressions at the sides of the neural plate could not be distinguished before the appearance of the neural crests. With the thickening of the ectoderm to form the crests, these structures are slightly elevated and a lateral linear* depression is visible, not only on the median, but often also on the lateral side of the neural crest. The neural ridges increase in size and length, growing anteriorly, posteriorly, and dorsally until they become continuous in front of and behind the neural plate. As the neural crests grow dorsally, the anterior part of the neural fold rises prominently, and the embryo has the appearance shown in figure 1. At this stage the blastopore has closed to a narrow sHt and the neural groove extends from the blastopore almost to the anterior part of the neural fold. On either side, just caudal to and within this anterior part of the fold, there is visible occasionally a small depressed circular or oval deeply pigmented area. These depressions were described by Eycleshymer, and, according to him, are the initial stages of the paired eyes.
In many of the embryos a few (usually three to five) faint transverse grooves appear in the anterior part of the medullary plate, but they are not constant in number or regular in position.
In some specimens other similar divisions appear posterior to these, but they are less distinct and gradually fade out posteriorly so that their number could not be determined. Normally, the transverse grooves first appear at the lateral edges of the plate and extend toward the neural groove. Frequently one appears on either side before the others and since these first ones are at a corresponding level, their fusion forms a furrow which I regard as the transverse cephalic groove of Griggs. There is considerable irregularity in the formation of the grooves, however; often those of the two sides are formed at different levels and do not meet at the median line. The areas between the grooves are then irregular in size and shape; frequently they are almost triangular as the transverse grooves converge or meet, either at the neural groove or at the lateral edge of the plate. The transverse grooves in the two sides of the neural plate of the same embryo do not regularly correspond, and this lack of correspondence is manifest in the figures of Griggs and other authors. Only in the occasional and unusual specimen is there present the regular arrangement described by Griggs. The neural folds close rapidly and it is possible to observe the changes that take place during the process. It is a significant fact that the grooves do not always retain precisely their original aspect during the closing process. Some of the grooves shift slightly or fade out entirely and other grooves appear in different positions.
Divisions of the neural plate caused by the transverse grooves could not be clearly or satisfactorily demonstrated in sections, but such study shows that, with the appearance of the transverse grooves, the mesoderm has developed to a stage where it is assuming a segmented condition, and I regard the formation of the transverse grooves as due to the formation of the mesodermal somites. I am convinced that certain of the transverse grooves coincide with the divisions between somites, and I am inclined to beheve that it is true of most if not all of the transverse grooves. It is possible, however, that grooves are also due to associated mechanical factors, pressure produced by the multiplying cells and the infolding of the neural crests.
In the lateral ridges a beaded appearance is sometimes present, but in no case did it show the regularity described by Locy. The lobulations along the neural crests are often entirelj^ absent, and, when present they do not show definite regularity, either in size or arrangement. The number of lobes varies from two or three to as many as fifteen on one side, and little if any correspondence could be detected between the lobulations of opposite sides of the same embryo. Sections of the crests show the cells to be distributed uniformlj^ with occasional slight irregular groupings, but there is no evidence of a segmental arrangement. These aggregates or clusters of embryonic cells are not differentiated into regular areas, but appear to be centers of rapid cell proliferation. Before the neural folds close the forebrain and midbrain are clearly outlined by thickened enlargements, and by the time of complete fusion, the three primary brain vesicles are distinctly defined. The crests close rapidly and in essential respects these observations confirm the description given by Eycleshymer ('95). The median divisions disappear with the closure of the neural folds, and no definite relation between them and the brain vesicles could be determined. Sections of many embryos seem to indicate that their fate is not uniform, but differs in different individuals.
For the study of chick embryos, several hundred eggs were incubated, and over one hundred embryos were obtained for study, giving a series of stages from the formation of the primitive streak to the formation of the brain and spinal cord. Most of the embryos were removed at the stage when the neural groove is open, as this, according to Locy and Hill, is the most favorable period at which to observe the primary neuromerism of the nervous system. In the study of the living embryo most of the work was done with a binocular although both dissecting and compound microscopes were used. For illumination, transmitted light, as well as reflected light from an electric arc, a gas light, and also direct sunlight were used. Following Locy's suggestion that "a dead black background is of course the best surface for observing anything of this kind by reflected light," a circle of dead black paper was placed under the specimen in the bottom of the watch-glass. The neural tube was swept clean of all surrounding tissues by the use of dissecting needles and fine brushes, the sides of the neural tube were dissected and also sections were cut of the roof and floor. In order to get shadows, specimens were tilted and rotated to secure all angles and degrees of illumination. Kleinenberg's, Bouin's, and Gilson's killing fluids were used, and some of the embryos were examined after faintly staining them with borax carmine and Conklin's picro-haematoxylin. To supplement the study of whole and dissected specimens, sections were cut in transverse, frontal, and sagittal planes and stained with Ehrlich's acid haematoxylin and Heidenhain's iron haematoxylin.
Figs. 1 to 7 and 12 to 14, camera-lucida drawings of Amblystoma embryos, showing successive stages of development, the so-called primary neuromeres, and the neuromeres of later stages.
Fig. 1 Early stage in the formation of the neural folds, showing the anterior thickening.
Fig. 2 Embryo showing neural groove, the lobulations along the neural folds, and transverse grooves of the neural plate.
Fig. 3 Embryo with no evidence of segmentation in the neural folds and regular transverse grooves of the neural plate.
Fig. 4 Embryo showing irregular character of the divisions of the neural folds and neural plate.
Fig. 5 Embryo showing expanded anterior part of the neural plate, with irregular divisions of the neural folds and neural plate.
Fig. 6 Embryo elongated, with partial fusion of the neural crests.
Fig. 7 Complete fusion of neural crests and appearance of the brain vesicles.
Fig. 12 Frontal section through the open neural crests of an embryo, showing irregular lobulations of crests and indefinite cell arrangement.
Fig. 13 Frontal section through the developing brain vesicles of embryo, showing the later neuromeres.
Fig. 14 Cross-section of embryo at same state of development as figure 13, showing mesodermal segmentation.
No indication of anything that could be interpreted as segmentation could be observed in the primitive streak, or before the neural folds were clearly outlined as elevated ridges. At this stage along the elevated margins of the medullary plate certain lobulated irregularities are formed, giving a beaded appearance to the crest, and these structures are present with more or less uniformity in most embryos up to the closure of the neural tube. They are, however, irregular in number in different embryos and do not correspond in the two sides of the same individual; their limits are often so obscure that they cannot be determined with certainty, and the wide variation in their relative position makes it impossible to correlate them, either in the two sides of the same embryo or in different embryos. They differ greatly in size; often in the same embryo there are two or three on one side while the corresponding region of the opposite side will consist of a single lobe, or perhaps the edge will be smooth, showing no lobulation. The variation in size, together with the uncertain position and desultory arrangement suggest strongly that this marginal lobulation is due entirely to differences in rate of cell proliferation at different points along the rapidly expanding wall of tissue.
As the neural crests increase in size, they rise rapidly and begin to fold over toward the median plane. At this stage especial care was exercised to detect any indication of segmentation in the medullary folds or in the plate between the folds.
Occasional faint lines could be distinguished, but their position and appearance were so irregular and variable that no segmental importance could be attached to them.
Segmentation of the neural folds was reported by Hill ('00), who described the marginal segments as separated by constrictions that in early stages completely encircle the encephalon, and in later stages are confined to its base and lateral walls." In the examination of large numbers of embryos, I have found on the external surface of the neural folds faint constrictions that appear as lines when the best shadow effects are obtained, but I fail to find the regularity described and figured by -Hill. On the contrary, the grooves are irregular in number and position, and often a single groove will divide to form two. The grooves do not regularly encircle the encephalon, but a groove will often fade out at some point and slightly anterior or posterior to it another groove will appear, so that the number of constrictions is different for the two sides. These constrictions are so faint that they can be traced only with difficulty, and in no specimen approxunate the condition shown in Hill's figures. Furthermore, dissection shows that internal grooves do not regularly correspond with external constrictions. I find external grooves are present at these stages with no corresponding internal constrictions and internal grooves with no corresponding external constrictions. Later in ontogenj^ Hill says the internal grooves are elevated upon the apices of internal ridges, but the groove at the apex of the internal ridge is not present with sufficient constancy to be of value in determining the constrictions that are of segmental importance. That there are grooves in addition to those considered by Hill to be segmental, he admits when he says, page 423, secondary divisions that frequently are present would eventually be confused with the primary ones." But he gives ho criteria by which to distinguish between primary and secondary constrictions, and in his figures certain ones are exaggerated as 'primary,' while others are suppressed as 'secondary.' He states that in these early stages the only criteria by which he determined segments are the external and corresponding internal grooves, and that he considers the internal ridge as a secondary modification. Observation of a large number of embryos affords no evidence to support the statement of Hill that eleven constrictions are present on both inner and outer surfaces of the open neural groove, that they are constant in number and nearly equal in size and that they appear earher in ontogeny than the historic encephahc divisions, forebrain, midbrain, and hindbrain." Hill reported that the third and fifth grooves are deeper than the others and mark the posterior limits of the forebrain and midbrain. While it is true that with the appearance of the so-called secondary division of the neural tube into forebrain, midbrain, and hindbrain, the limits that separate them are clearly marked, the present study fails to confirm his statement "that an earlier segmentation is incorporated into this division as follows: in the forebrain three primarj^ somites; in the midbrain two; and in the hindbrain, six or six and one half if the portion of segment twelve that lies in front of the first somite is added to the latter."
Figs. 8 to 11 and 15 to 20, camera-lucida drawings of chick embryos, showing successive stages of development, the so-called primary neuromeres and the neuromeres of later stages.
Figs. 8 and 9 Embryos of three somites, dorsal view, drawn from living specimens, showing the early beaded appearance of the neural crests.
Figs. 10 and 11 Embryos of eight and thirteen somites, respectively, dorsal view, drawn from living specimens, showing the brain vesicles and later neuromeres.
Fig. 15 Frontal section through the neural crests of an embryo of three somites, showing the irregular lobulations of the crests, absence of corresponding external and internal grooves, and indefinite cell arrangement.
Fig. 16 Frontal section through the right neural crest of an embryo of five somites, showing same features as figure 15.
Fig. 17 Sagittal section through the closing neural tube of an embryo of ten somites, showing same features as figure 15.
Fig. 18 Sagittal section of the floor of medullary tube of an embryo of five somites, showing same features as figure 15.
Fig. 19 Frontal section through the developing brain vesicles, showing the later neuromeres.
Fig. 20 Same section as figure 19, showing cell arrangement in the right wall of the hind brain just anterior to the otic invagination.
As the neural crests approach each other, the primary bram vesicles and neuromeres of the hindbrain are well defined, and fusion of the folds first occurs in the region of the midbrain vesicle or slightly posterior to it. After the closure of the neural tube there are clearly six segments anterior to the auditory invagination. These brain vesicles and neuromeres of the hindbrain are so well-known that further description is unnecessary. In these divisions there is present the definite, characteristic cell arrangement designated by Orr as distinguishing true neuromeres. In the open neural groove there is no arrangement of cells in the medullary ridges or floor that even suggests a segmental condition. The cells are evenly distributed and do not manifest any tendency toward groupings that would give morphological significance to the grooves which serve as the basis of the primary neuromerism of Locy and Hill.
A survey of the literature on the subject of head segmentation shows most unusual differences, both in observation and interpretation. These observations are based on the study of different morphological features, and it has been impossible satisfactorily to explain the discrepancies and differences reported. In the study of neuromerism in urodeles, Kupffer, Froriep, Eyclesh^mer, Neal, Locy, Griggs, Smith, and others have described as many as eleven and as few as three segments in the cephalic region. Locy considered the divisions of the neural crests as segmental, and did not regard the divisions of the cephalic plate as of metameric importance. Kupffer, Froriep, Eycleshymer, Griggs, Smith, and others have agreed that in any consideration of neuromeric segmentation, the divisions of the medullary plate are of primary importance, but these authors have not agreed as to their number or metameric significance, and most regard them as due to the segmentation of the mesoderm. Locy pointed out that the median divisions do not correspond with those in the neural ridges; he reports four or five divisions in the median plate and ten or eleven segments in the neural ridges of the same region. He says, page 530, Whether we find the median plate smooth in Amblystoma or faintly segmented depends on the stage at which the examination is made, and we recognize that the appearances in any one egg are not constant throughout the open groove stage; further, that eggs of closely related animals are by no means necessarily similar at corresponding stages." According to Griggs, the beaded appearance of the neural crests was not apparent in any of the embryos of this stage examined by him, and he argues for the metameric significance of the median divisions. Locy and Griggs both attempt to establish neuromerism as a basis for determining the segmentation of the head, but their results are mutually exclusive and contradictory.
Locy's statement that primary neuromeres are visible in the blastoderm of the chick at the twelfth hour of incubation, just as the head fold is first outlined, and that they extend into the primitive streak, finds absolutelj^ no support in any of thematerial of the present investigation. His further statement that the cells in these segments are characteristically arranged, even in the earhest stages, and their arrangement and structure would indicate that they are definite differentiations of cell areas" was denied by Hill, and in the present study evidence to support this statement of Locy is also entirely wanting. Neal ('98) called attention to the fact that "none of the reproductions of Locy's photographs, with two possible exceptions, show a segmentation of the neural folds in either the trunk or embryonic rim." He might well have added that none show neuromeres of both sides and that the same embryo was not photographed twice, in two different positions (which probably would be necessary) that the neuromeres of the two sides might be compared. In fact, Locj^'s photographs, in my opinion, deny rather than confirm his statement. He admits that his drawings "are a little too distinct" and "the exactness has been exaggerated." Hill's figures of the chick have called forth exclamations of surprise and astonishment on all sides. He figures constrictions of his primary neuromerism persisting in embryos with a closed neural groove, but I have been unable to observe such a condition. It is a significant fact that the cell arrangement of the forebrain and midbrain at this stage does not show such segmentation. In the primary neuromeres, he admitted that the segmental arrangement of cells is" absent, and argued that the radial cytological condition which later appears is due to the intrasegmental expansion of the walls. Thus the definite structure of the later neuromeres he attempted to explain on purely mechanical grounds. If, however, these definite and constant structural features be merely mechanical effects, one wonders how he can hope to substantiate the transitory, indefinite, and irregular headings of the early stages as a primary neuromerism of phylogenetic importance.
Study of the neuromeres of the later stages has also led to great diversity of opinion. The neuromeres of the medulla are definite structures with characteristic morphological features. It is an open question whether or not they are homologous with the divisions of the neural tube anterior and posterior to them. The divisions of the spinal cord are undoubtedly formed by the pressure of the adjacent mesodermic somites, and anterior to the otic invagination the number and character of the somites are far from established. It is in the anterior part of the neural canal that the evidence is most scanty and indefinite. Neal ('18) argues that the primary brain vesicles are the true neuromeres of the region, and in the opinion of the writer his argument is clear and comprehensive. If the central nervous system of the primitive vertebrate were segmented, with the enlargement of the brain there would be an enlargement of the segments. The brain segments enlarge laterally and dorsoventrally, and it seems only natural that they should enlarge anteroposteriorly. It certainly is as reasonable to suppose that individual segments would expand anteroposteriorly as to account for the elongation of the brain by fusion of segments and the backward migration of the cephalic region with the concomitant incorporation of additional segments in the brain. While gill clefts, visceral arches, and epibranchial organs are cenogenetic, still they may be segmental, being predetermined in position by nerves, blood vessels, septa, and other segmental structures of the invertebrate. The later brain is highly developed, with great specialization of parts, and ontogeny affords such fragmentary and inconclusive evidence of phylogeny that neuromerism alone can hardly explain the development of the head. The study of highly specialized forms like the chick must appear of less importance than that of more primitive forms, or at least of forms in which primitive conditions persist. In this connection, Xeal ('18) has pointed out that neuromeres are more conspicuous in the embryos of higher, than they are in embryos of lower chordates, and this would hardly be expected if they are vestiges of a primitive neuromerism.
In ontogeny, segmentation regularly appears first in the mesoderm and the segmentation of the mesoderm is more constant and regular than segmentation in other tissue. Segmentation of other tissue normally results from and is in correspondence with segmentation of the mesoderm. Mesomeres are uniformly present in the lower chordates, and to disregard mesodermal segmentation is therefore to overlook an item of paramount importance in any study of head segmentation. In the ancestral vertebrate there was undoubtedly a correspondence of mesomeres, neuromeres, cranial nerves, and branchial organs, and all of these structures must be considered in an explanation of the present lack of correspondence.
The present study has shown that in Amblystoma and the chick at least, the structures described by Locy and Hill as primary segments cannot be regarded as metameric. Investigators have repeatedly questioned the accuracy of the observations of Locy and Hill, and a repetition of their work, using as far as possible identical means of examination, has in the present case not only failed to verify their observations, but disclosed a quite different condition. The three morphological features upon which neuromerism can be based, marginal headings, external and internal grooves, and cell arrangement, all fail to give evidence to confirm the primary neuromerism of Locy and Hill. Neal could not confirm Locy's statements concerning Selachian embryos and I have been unable to confirm Locy's observations on Amblystoma or Hill's on chick embryos. In my opinion, the so-called 'primary metamerism' is based upon incorrect observation and cannot be accepted. The median divisions observed in the neural plate of Amblystoma are largely if not entirely due to segmentation of the mesoderm, and so can be regarded onlj^ as features of secondary importance. The primary neuromeres of Locy and Hill, as well as those of Griggs and other students of neuromerism, are irregular in size, inconstant in number, asymmetrical in position, and cannot serve as trustworthy criteria of the metamerism of the vertebrate head.
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